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- Kardeby, Caroline, 1989-, et al.
(författare)
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Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα
- 2019
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 3:3, s. 275-287
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Tidskriftsartikel (refereegranskat)abstract
- Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.
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- Fälker, Knut, 1971-, et al.
(författare)
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Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation
- 2019
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 59, s. 96-109
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Tidskriftsartikel (refereegranskat)abstract
- The healthy vascular endothelium constantly releases autacoids which cause an increase of intracellular cyclic nucleotides to tame platelets from inappropriate activation. Elevating cGMP and cAMP, in line with previous reports, cooperated in the inhibition of isolated human platelet intracellular calcium-mobilization, dense granules secretion, and aggregation provoked by thrombin. Further, platelet alpha granules secretion and, most relevant, integrin αIIaβ3 activation in response to thrombin are shown to be prominently affected by the combined elevation of cGMP and cAMP. Since stress-related sympathetic nervous activity is associated with an increase in thrombotic events, we investigated the impact of epinephrine in this setting. We found that the assessed signalling events and functional consequences were to various extents restored by epinephrine, resulting in full and sustained aggregation of isolated platelets. The restoring effects of epinephrine were abolished by either interfering with intracellular calcium-elevation or with PI3-K signalling. Finally, we show that in our experimental setting epinephrine likewise reconstitutes platelet aggregation in heparinized whole blood, which may indicate that this mechanism could also apply in vivo.
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| 4. |
- Grenegård, Magnus, 1963-, et al.
(författare)
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The cardioprotective, anti-inflammatory and antithrombotic piperazinyl-purine analogue MK177 is a bifunctional drug with promising therapeutic potential
- 2023
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Ingår i: British Journal of Pharmacology. - : Macmillan Publishers Ltd.. - 0007-1188 .- 1476-5381. ; 180:Suppl. 1, s. 158-159
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Nitrate ester bearing 6-piperazinyl-purine analogues (denoted MK drugs) are cardioprotective in infarction animal models and act as inhibitors of Janus kinase (JAK) and Rho-associated kinase (ROCK) [1-3]. Despite the presence of nitrate ester moiety, the MK drugs do not release nitric oxide (NO).Methods: We utilized organic chemistry platforms to design a dinitrate ester derivative denoted MK177, cell-free and cellular assays to elucidate antithrom-botic and anti-ischemic mechanisms. Furthermore, we also used tissue models to analyze vasodilation, and animal models to evaluate drug activities in vivo.Results: In anesthetized pigs, intravenous infusion of MK177 produced“nitroglycerin-like”effects on vital parameters. Analysis of exhaled air confirmed release of NO. MK177 caused concentration-dependent relaxation of iliac arteries (87±6.8 % relaxation of precontracted arteries, mean value ±SD, n=5) and this effect was mediated by activation of the NO/cyclic GMP signaling pathway. It is noteworthy that other mononitrate or non-nitrate MKs did not cause NO-induced vasodilation. In cellular model systems, MK177 evoked antithrombotic effects by targeting ROCK in a NO-independent manner. Specifically, MK177 inhibited platelet aggregation induced by collagen (72±12.6 % inhibition of aggregation, mean value±SD, n=7). Western blot analyses confirmed that MK177 reduced ROCK-dependent phosphorylation of myosin phosphatase sub-unit (MYPT-1) in platelets. Finally, kinase screening assay revealed that MK177 concentration-dependently inhibited ROCK and JAK (Kd values around 5μM).Conclusion: We have developed a bifunctional drug molecule, MK177, that acts by NO-dependent and NO-independent mechanisms. MK 177 induced car-diovascular NO effects in vivo and relaxed vessels in vitro. MK177 also prevented blood platelet activation via NO-independent ROCK inhibition. The bifunctional nature of MK177 can be of significance in future management of thrombotic and ischemic disease. Collectively, the novel cardio-protective and bifunctional drug MK177 has promising therapeutic potential.References:1. Koufaki M, Fotopoulou T, Iliodromitis EK, Bibli SI, Zoga A, Kremastinos DT, Andreadou I. Discovery of 6-[4-(6-nitroxyhexanoyl)(piperazin-1-yl)]-9H-purine, as pharmacological post-conditioning agent. Bioorg Med Chem. 2012;20(19):5948-5956. https://doi.org/10.1016/j.bmc.2012.07.0372. Kardeby C, Paramel GV, Pournara D, Fotopoulou T, Sirsjö A, Koufaki M, Fransén K, Grenegård M. A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition. Eur J Pharmacol. 2019;15(857):172428-172434. https://doi.org/10.1016/j.ejphar.2019.1724283. Paramel GV, Lindkvist M, Idosa BA, Sebina LS, Kardeby C, Fotopoulou T, Pournara D, Kritsi E, Ifanti E, Zervou M, Koufaki M, Grenegård M, Fransén K. Novel purine analogues regulate IL-1βrelease via inhibition of JAK activity in human aortic smooth muscle cells. Eur J Pharmacol. 2022;15(929):175128-175135. https://doi.org/10.1016/j.ejphar.2022.175128
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- Lindkvist, Madelene, 1984-
(författare)
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Impact of Interleukin-6 family cytokine signalling on human endothelial cells and platelets
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Endothelial cells lining the luminal side of blood vessels creates a barrier between the circulating blood and the extracellular matrix. Endothelial cells have important functions in regulation of vessel tension and inflammation. Furthermore, endothelium-derived vasodilators prevent our smallest blood cells, platelets, to aggregate in the circulation. The main physiological role of platelets is to protect us from bleeding by creating aggregates at sites of injury. Platelets is also increasingly recognised as mediators in acute inflammation. The focus of this thesis has been to study the impact of inflammatory cytokines in the interleukin (IL)-6 family on endothelial cells and platelets. IL-6 has pleiotropic effects where IL-6 trans-signalling via the soluble IL-6 receptor (IL-6R) is associated to more pro-inflammatory outcomes than classic signalling via the membrane bound IL-6R. Both classic and trans-signalling need the ubiquitously expressed glycoprotein (gp)130 to induce intracellular signalling. Since the IL-6R is expressed on a restricted number of cell types, transsignalling exerts a broader IL-6 response. Paper I reveal that endothelial cells express IL-6R which facilitates both classic and trans-signalling. IL-6 trans-signalling activates more signalling pathways and results in proinflammatory responses in contrast to classic signalling. Paper II show that IL-6 trans-signalling, but not classic signalling occurs in platelets and results in inhibition of epinephrine-induced platelet aggregation. Paper III reveal inter-individual differences in platelet reactivity towards activators and the inhibitor nitric oxide (NO). Individuals with more NOsensitive platelets showed greater capacity of vasodilation, indicating a connection between endothelial function and platelet inhibition. In Paper IV, the impact of various gp130 signalling cytokines on endothelial cells revealed differences and similarities in intracellular signalling, gene expression and protein release. In summary, this thesis investigates the impact of the IL-6 family cytokines on endothelial cells and platelets in regards of intracellular signalling and functional responses.
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| 10. |
- Lindkvist, Madelene, 1984-, et al.
(författare)
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Individual variations in platelet reactivity towards ADP, epinephrine, collagen and nitric oxide, and the association to arterial function in young, healthy adults
- 2019
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Ingår i: Thrombosis Research. - : Elsevier. - 0049-3848 .- 1879-2472. ; 174, s. 5-12
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Platelet aggregation and secretion can be induced by a large number of endogenous activators, such as collagen, adenosine diphosphate (ADP) and epinephrine. Conversely, the blood vessel endothelium constitutively release platelet inhibitors including nitric oxide (NO) and prostacyclin. NO and prostacyclin are also well-known vasodilators and contribute to alterations in local blood flow and systemic blood pressure.MATERIALS AND METHODS: In this study we investigated individual variations in platelet reactivity and arterial functions including blood pressure and flow-mediated vasodilation (FMD) in 43 young, healthy individuals participating in the Lifestyle, Biomarkers and Atherosclerosis (LBA) study. Platelet aggregation and dense granule secretion were measured simultaneously by light transmission and luminescence. FMD was measured with ultrasound.RESULTS: The platelet function assay showed inter-individual differences in platelet reactivity. Specifically, a sub-group of individuals had platelets with an increased response to low concentrations of ADP and epinephrine, but not collagen. When the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) was combined with high doses of these platelet activators, the results indicated for sub-groups of NO-sensitive and NO-insensitive platelets. The individuals with NO-sensitive platelets in response to SNAP in combination with collagen had a higher capacity of FMD of the arteria brachialis.CONCLUSIONS: Platelet reactivity towards ADP, epinephrine and NO differs between young, healthy individuals. Some individuals have a more effective response towards NO, both in the aspect of platelet inhibition ex vivo, as well as vasodilation in vivo.
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